Heterogeneous distribution of chloride channels along the distal convoluted tubu - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:08 |只看该作者 |倒序浏览 |打印

作者：Antoine Nissant, Stéphane Lourdel, Sophie Baillet, Marc Paulais, Pedro Marvao, Jacques Teulon, and Martine Imbert-Teboul作者单位：Centre National de la Recherche Scientifique and Université Pierre et Marie Curie, UMR 713 Institut des Cordeliers, 75270 Paris Cedex 0 France



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      【摘要】
   The distal convoluted tubule (DCT) is a heterogeneous segment subdivided into early (DCT1) and late (DCT2) parts, depending on the distribution of various transport systems. We do not have an exhaustive picture of the Cl - channels on the basolateral side: the presence of ClC-K2 channels is generally accepted, whereas that of ClC-K1 remains controversial. We used here single-cell RT-PCR and patch clamp to probe Cl - channel heterogeneity in microdissected mouse DCT at the molecular and functional levels. Our findings show that 63% of the DCT cells express ClC-K2 mRNA, either alone (type 1 cells: 47 and 23% in DCT1 and DCT2, respectively), or combined with ClC-K1, mostly in DCT2 (type 2 cells: 33%), but 37% of DCT1 and DCT2 cells do not express any ClC-K. Patch-clamp experiments revealed that a Cl - channel, with 9-pS conductance and Cl - NO 3 - = Br - anion selectivity sequence, is present in the DCT1 and DCT2 basolateral membranes (87 and 71% of the patches, respectively). This dominant channel is likely to be ClC-K2 in type 1 cells. In type 2 cells, it could be ClC-K2 and/or ClC-K1 homodimers, but also ClC-K1/ClC-K2 heterodimers, or a mixture of all combinations. A second, distinct Cl - channel (13% of DCT1 patches, 29% of DCT2 patches) also displayed 9-pS conductance but had a completely different anion selectivity (I - NO 3 - Br - Cl - ), which was not compatible with that of the ClC-Ks. This indicates that a Cl - channel that is unlikely to belong to the ClC family may also be involved in Cl - absorption in the DCT2. - v  ]6 F$ e! w' S( Q; d. G
      【关键词】 kidney ClCK sodium chloride absorption5 J# v5 a0 \) @2 n% X+ r4 s( s" R5 O
   IT HAS LONG BEEN ACCEPTED that the distal convoluted tubule (DCT) of the mammalian kidney is homogeneous at the cell level, consisting of a predominant cell type that reabsorbs sodium, calcium, magnesium, and chloride and, in its terminal portion, of a small number of intercalated cells that are involved in controlling the acid-base balance. It has now been established, however, that in most species, except the rabbit, the DCT is heterogeneous in terms of both cell structure and ion transport protein localization ( 25 ). For instance, the apical and basolateral transport systems implicated in calcium reabsorption are mainly located in the terminal part of the DCT (DCT2) ( 5, 6, 16, 17 ), whereas the main apical NaCl entry pathway, the thiazide-sensitive Na -Cl - cotransporter (NCC), is found to a greater extent in its initial portion (DCT1) than in DCT2 ( 16, 17 ). In addition, amiloride-sensitive sodium channels have been found at the luminal membrane of DCT2 cells but not that of DCT1 cells ( 16, 17 ). At the basolateral membrane, Na is extruded via the Na -K -ATPase, and Cl - is thought to leave the cells via a Cl - -selective conductance ( 25 ). So far, however, we do not have a clear picture of the chloride channels underlying this conductance, nor do we know whether they are evenly distributed in both parts of the DCT.8 `' j9 v' z2 H9 D2 Q( F0 l
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Patch-clamp experiments have detected the presence of a small-conductance Cl - channel at the basolateral membrane of the DCT1 ( 19 ), but the DCT2 has not been investigated. Three lines of evidence suggest that this channel might be ClC-K2, a kidney-specific member of the ClC Cl - channel family ( 13 ). First, mutations inactivating ClC-Kb, the human ortholog of ClC-K2, induce a variant of the Bartter syndrome that, in some patients, displays clinical evidence of DCT dysfunction ( 27, 39 ). Second, immunofluorescence studies have established that ClC-K2 is expressed in the DCT basolateral membrane ( 15 ). Third, the Cl - channel in the early DCT ( 19 ) exhibits the same sensitivity to extracellular calcium and pH as human ClC-K channels expressed in Xenopus laevis oocytes, and its anion selectivity is compatible with that of ClC-Kb ( 8, 35, 36 ).' U) U) x: ?. T4 R, b& o$ `3 s5 X4 _7 V
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These data are convincing but do not rule out the possibility that other channels may also contribute to the process of distal Cl - absorption. Calcium-dependent Cl - currents have been detected in primary cultures of DCT cells using a whole cell recording method ( 2 ). However, their apical or basal origin was not determined. Nor was it established whether they are expressed in the native tissue. Another likely candidate is ClC-K1, a channel highly homologous to ClC-K2, and which is also expressed in the kidney. The possible presence of ClC-K1 in cortical nephron segments has long been a matter of debate ( 32, 33, 38 ), but we recently showed that the corresponding mRNA is expressed, as is that of ClC-K2, in the mouse DCT ( 19 ). Due to the high level of homology, attempts to generate specific antibodies for each ClC-K have so far been unsuccessful. ClC-K1-deficient mice have been used to localize ClC-K2 along the renal tubule ( 15 ), but ClC-K2-deficient mice are not available, which means that it is impossible to investigate ClC-K1 using immunofluorescence. Moreover, although both mRNAs are expressed in the DCT, we do not know whether they are located in the same cells. If they are located in the same cells, channels may be formed not only as ClC-K1 or ClC-K2 homodimers but also as ClC-K1/ClC-K2 heterodimers, as has already been reported for other ClCs expressed in X. laevis oocytes ( 18, 37 ).( m3 `2 k( K3 e! R2 Q

On the basis of these findings, we used a single-cell RT-PCR method to analyze the distribution of ClC-K1 and ClC-K2 mRNAs along the mouse DCT. Because ClC-K1 and ClC-K2 mRNAs were coexpressed in some, but not all, DCT2 cells, separate patch-clamp experiments were used to investigate the properties of Cl - channels in DCT2. Our results reveal that the molecular and functional heterogeneity observed in the DCT for a series of ion transport systems also includes the expression of Cl - channels.
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METHODS
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Animals and Tissue Preparation

The experiments were conducted according to the rules of the French Ministry of Agriculture (license no. 7427) in male CD1 mice (Charles River, l'Arbresle, France), which were fed the regular laboratory chow ad libitum (SAFE, Epinay, France). Animals were killed by cervical dislocation. One kidney was perfused with an L-15 Leibovitz medium (Sigma, Saint Quentin Fallavier, France) supplemented with collagenase (Worthington CLS II, 300 U/ml) before being removed. Small pieces of the cortex and medulla were then incubated in the same medium at 37°C for 45-60 min, rinsed, and kept at 4°C ( 19 ).  \1 i* e  m; [$ L, L

For the RT-PCR experiments on tubular RNA extracts, fragments of the cortical thick ascending limb of Henle's loop (CTAL), distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD) were microdissected in the perfusion solution supplemented with BSA (1 mg/ml) at 0-4°C. They were then rinsed in fresh medium and either directly processed for RNA extraction ( 7 ) or permeabilized with Triton X-100 and kept at -80°C until reverse transcription was performed.5 P0 g0 S4 o1 B4 `7 M/ P

For the patch-clamp and single-cell RT-PCR experiments, nephron fragments including the DCT and the end of CTAL were isolated and directly transferred into the superperfusion chamber. The site of patch clamping was located using a graduated scale placed in one of the microscope eyepieces, taking the abrupt transition between the CTAL and DCT as the origin. Patch-clamping sites in the early portion of DCT (referred to as DCT1) were located from 46 to 192 µm from the end of the CTAL (mean distance: 99.4 ± 9.3 µm, n = 20), and those in the late portion (DCT2) from 320 to 558 µm from the end of CTAL (mean distance: 412.4 ± 16.5 µm; Fig. 1 ).8 d7 C# S5 |% ?4 Q! Y1 H+ N

Fig. 1. Representative example of mouse distal convoluted tubule (DCT), microdissected from a collagenase-treated kidney, as described in METHODS. Thick arrows indicate the DCT boundaries, i.e., from the end of cortical thick ascending limb (CTAL), after macula densa (md), to the beginning of connecting tubule (CNT; identified here by a decrease in the tubule outer diameter). The whole length of this DCT (DCT1 2), as measured by image analysis, was 560 µm, a value representative of the mean length, calculated from similar measurements in several mice (580 ± 20 µm, n = 13). Because the transition between DCT1 and DCT2 cannot be easily distinguished, the sites of electrical recordings and cytosol sampling (dotted rectangles) were restricted to the initial and terminal portions of the DCT, i.e.,

Real-Time PCR
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Reverse transcription was performed on 8-µl tubular RNA extracts using the first-strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Meylan, France), according to the manufacturer's protocol. The remaining 2 µl of the RNA extract were processed in parallel without reverse transcriptase and served as negative controls (RT - )./ E; O- T: R+ s4 y: {

Real-time PCR was performed on a LightCycler (Roche Diagnostics) with the LightCycler FastStart DNA Master SYBR Green 1 kit (Roche Diagnostics) according to the manufacturer's protocol, except that the final reaction volume was reduced to 8 µl. PCR was performed in the presence of a quantity of cDNA corresponding to 0.1 mm of nephron, or of similar dilutions of the RT - controls. Samples were subjected to 42 cycles of three temperature steps (95°C for 10 s; 61-64°C for 10 s; and 72°C for 15 s), followed by a fusion curve to check for the absence of contaminating PCR products. After 42 cycles, no DNA was detectable in the RT - samples or in blanks run without cDNA. In each experiment, a standardization curve was plotted using serial dilutions (1 to 1/500) of a standard cDNA stock solution made from whole kidney RNA ( dilution 1 corresponded to the cDNA produced from 10 ng RNA). For all the samples, the amount of PCR product was calculated as a percentage of the RNA standard per millimeter of tubule length (arbitrary unit). Data were then corrected for PCR efficiency, and the relative expression of two genes (1 and 2) in a given sample was calculated from the following equation: N 1 / N 2 = E 2 CP2 /E 1 CP1. L 2 / L 1, where N 1 and N 2 represent the number of cDNA copies present in the sample before PCR amplification; E 1 and E 2, the PCR efficiencies; CP 1 and CP 2, the crossing points (number of cycles corresponding to the detection threshold of the PCR products fluorescence); and L 1 and L 2, the respective lengths of the amplified fragments. Assuming that the reverse transcription efficiency was similar for all mRNAs, N 1 / N 2 provides a good estimate of the relative expression of genes 1 and 2 in the extract. For each sequence and each mouse, PCR efficiencies and crossing points were measured in at least four different experiments, and the mean values were used to calculate the N 1 / N 2 ratio.5 {: p3 l: G0 L3 J( g& ~; [
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Single-Cell RT-PCR4 U7 p+ v& v" z4 B! }$ e
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Cytosol collection. Patch-clamp pipettes were made just before use from thoroughly cleaned borosilicate glass (GC150T, Harvard Apparatus, Edenbridge, Kent, UK), coated with Sylgard, and filled with 8 µl of solution. The solution used to fill the pipette was made from RNAse-free chemicals, filtered through a 0.22-µm filter, and stored at -20°C. It contained (in mM) 145 CsCl, 1 CaCl 2, 1 MgCl 2, and 10 HEPES (pH 7.4). After a gigaseal was obtained, suction was applied to the pipette to break the membrane patch, and the cytosol was harvested within the tip of the pipette. Cytosol samples and blanks (i.e., samples of peritubular medium collected beside the patched cell) were then treated in parallel for reverse transcription and PCR, as previously described ( 1 ).3 ^% w; H: @( Z9 W! S, Q# r

Reverse transcription. After the cytosol had been aspirated, the tip of the pipette was broken at the bottom of a test tube and its contents expelled into 2.6 µl of a mix containing 2 µl of 5 x medium {2.5 mM of each dNTP (Invitrogen, Paisley, UK) and 25 µM random primers [pd(N)6, Roche Diagnostics]}, 0.5 µl of 200 mM DTT, and 0.1 mM MgCl 2. RNAsin (0.5 µl, 20 U) and Superscript II reverse transcriptase (0.5 µl, 100 U, Invitrogen) were then added to each tube, and the RT reaction was processed at 37°C overnight. Because the expelled volume containing the cytosol was 6.5 µl, the final volume was 10 µl, with final concentrations of 0.5 mM for each dNTP, 5 µM for the random primers, 10 mM DTT, and 2.65 mM MgCl 2.

In some experiments, RT was carried out under the same conditions on Triton X-100-permeabilized tubules and tubular RNA extracts (all diluted in 5 µl of microdissection medium); 2 µl of the 5 x RT mix, 1.0 µl of 100 mM DTT, 0.1 µl of 200 mM MgCl 2, and 1.4 µl of water were added to each reaction tube. RT (final volume 10 µl) was initiated at room temperature by adding 0.5 µl RTase. After a preequilibration period of 10 min at 25°C, cDNA was synthesized at 42°C for 1 h. Negative RT controls were performed by omitting the RTase. The cDNA were stored at -20°C until PCR.3 J4 r) S6 p; {. x

PCR. Two PCR steps were sequentially carried out: during the first one (multiplex PCR1), six different cDNAs were coamplified in the same tube in a final volume of 100 µl. Seventy microliters of a PCR mix containing 2.5 U Taq DNA polymerase and 10 µl of 10 x PCR buffer (Qiagen, Courtaboeuf, France) were first added to the RT product (10 µl). All samples were kept for 1 min at 94°C before 20 µl of a mix containing the sense and antisense primers (10 pmol each) were added. After 1 min at 94°C, the samples were processed for 20 cycles at three sequential temperature steps: 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, with the exception of the last cycle in which the elongation phase lasted 10 min. The second PCR step (PCR2) was specific to each cDNA species. It was performed on 2-µl aliquots of PCR1 products. For this purpose, 78 µl of a PCR mix containing 10 µl 10 x Taq buffer, 2.5 U Taq DNA polymerase, and 1 µl of dNTPs (5 mM) were added to each tube; after the primers were added, the samples were processed as described above and subjected to 30 additional PCR cycles at the appropriate annealing temperature. PCR products were separated by electrophoresis on 2% agarose gels containing ethidium bromide and then viewed under UV light.: m' D4 e+ U$ h$ y" W5 l8 m+ ?0 G

Three different experimental series of single-cell RT-PCRs were carried out. In series I, the cDNA of the ClC-K1 and ClC-K2 channels were coamplified with those of the NaCl cotransporters NCC and NKCC2, the vasopressin V2 receptor Avpr2, and the -subunit of the amiloride-sensitive epithelial sodium channel -ENaC. In series II, NKCC2 was replaced by the calcium- and magnesium-binding protein parvalbumin (Pva), and -ENaC was investigated in some, but not all, experiments. Finally, in series III, NCC was replaced by tissular kallikrein (KlK6), a protein expressed in both DCT and CNT ( 23 ). In all these series, DCT1- and DCT2-cytosol samples were run in parallel in the same PCR experiments.

Primers
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Table 1 shows the primer sequences used for real-time and single-cell PCR. Preliminary experiments on whole kidney cDNA showed that only one PCR fragment of the expected size was detected for each of the genes. The identity of the amplified products was confirmed by digesting with restriction enzymes (data not shown). Using Triton X-100-permeabilized DCT and CNT segments, it was also shown that primers used in single-cell experiments for ClC-K1, ClC-K2, NCC, and -EnaC did not amplify genomic DNA, whereas primers for Avpr2 and KlK6 amplified two different DNA fragments, a short one (cDNA) in the presence of RTase, and a longer one (genomic DNA) in the absence of RTase (data not shown). The primers used for NKCC2 and Pva did not enable us to distinguish cDNA from genomic DNA. Before single-cell PCR experiments were initiated, tubular RNA extracts (amounts corresponding to 0.1-mm tubule length per tube) were used to check that all the sequences had been properly amplified, whatever the combination of primers.

Table 1. Primers4 r- n8 Z7 f/ }7 s5 m. e& p+ V" G4 s

Patch-Clamp Experiments# d  O0 s5 `- m. \
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In separate experiments, the cell-attached and cell-free configurations of the patch-clamp technique ( 11 ) were used on the DCT basolateral membrane. Currents were recorded using List LM-EPC7 or Bio-logic RK 400 patch-clamp amplifiers, monitored using Axoscope software (Axon Instruments, Foster City, CA), and stored on digital audio tape (Bio-logic, Claix, France). Stretches of current recordings were filtered at 300 or 500 Hz low-pass by an 8-pole Bessel filter (NPI Electronic, Tamm, Germany), digitized at a sampling rate of 1-2 kHz using a Digidata 1200 analog-to-digital converter, and analyzed with pClamp software (Axon). The tubules were initially bathed in physiological saline (PS) containing (in mM) 140 NaCl, 5 KCl, 1 CaCl 2, 1 MgCl 2, 10 glucose, and 10 HEPES (pH 7.4). The pipette solution used was similar, except that KCl was replaced by NaCl. The solutions used to characterize channel properties in the inside-out configuration contained 2 mM EGTA and no calcium. Anion vs. cation selectivity was tested using a low-NaCl solution in which the NaCl concentration was reduced to 14 mM (with 260 mM sucrose) and no KCl was added. For determining the anion selectivity, 130 mM NaCl on the bath side was replaced by an equivalent concentration of a sodium salt of the test anion. Potentials across cell-attached and excised membrane patches were corrected for liquid junction potentials ( 3 ). The experiments were carried out at room temperature (22-27°C).

Statistics
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Results are reported as means ± SE. Mean values in the different groups were compared using ANOVA with a protected least significance difference Fisher test.3 l& s1 w( C  ~+ _8 m
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RESULTS
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Molecular Identification of DCT Cells! a  v; [. _$ P& T4 P0 J: b
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As already mentioned, the DCT is a heterogeneous segment subdivided into two functionally different subsegments, DCT1 and DCT2 ( 16, 21, 25 ). The DCT1 is homogeneous at the cell level, but DCT2 involves intercalated cells in its terminal portion ( 21, 25 ). The boundaries between DCT1 and DCT2, and those between DCT2 and CNT, are progressive and not easily identified under the microscope ( Fig. 1 ). This made it necessary to check the identity of the patched cells a posteriori using PCR, by coamplifying different cDNA species (the cDNA of ClC-K channels and cDNA of cell markers) from the same cytosol sample. Based on functional and immunofluorescence data from the literature, DCT cells were defined as those that expressed NCC and Pva ( 6, 17 ) but neither NKCC2, the Na-K-2Cl cotransporter in the thick ascending limb ( 16, 26 ), nor Avpr2, the vasopressin receptor expressed in CNT and CCD, and to a lesser degree in TAL ( 20 ). In single-cell RT-PCR experiments, Pva and -ENaC were tentatively used as markers for DCT1 and DCT2 cells, respectively ( 6, 17 )., g( D5 h0 I. Q" p, a& I2 z

Figure 2 shows the expression patterns of NKCC2, NCC, Avpr2, Pva, and -ENaC mRNAs established on CTAL, DCT, CNT, and CCD extracts by real-time PCR ( Fig. 2 A ) and/or by the multiplex approach designed for single cells ( Fig. 2 B ). The mRNA distribution in all these segments matched that reported for the corresponding proteins ( 6, 17, 26 ). NKCC2 was occasionally detected in DCT, but the low and variable cDNA levels found in the different extracts suggested that this was attributable to contamination from adjacent CTAL cells, rather than to local mRNA expression.

Fig. 2. Distribution of Na-K-2Cl cotransporter (NKCC2), thiazide-sensitive Na -Cl - cotransporter (NCC), parvalbumin (Pva), -epithelial sodium channel (ENaC), and Avpr2 mRNA in distal segments of the mouse nephron. A : real-time PCR experiments carried out on CTAL, DCT, CNT, and cortical collecting duct (CCD) RNA extracts. Results (arbitrary unit per mm tubule length) are expressed as means ± SE from 3 animals. B : representative electrophoresis gel illustrating the results obtained in the same segments by the multiplex approach after 20 cycles PCR1 and 20-25 cycles PCR2, as described in METHODS. Note that a trace of NKCC2 (visible here in A and B ) was detected in the DCT, suggesting that some of the segments in the pool used for RNA extraction probably included some CTAL cells.8 O9 R. V- Y) d/ ]  Y' r  ]# s

ClC-K1 and ClC-K2 Expression in the DCT, ]: E5 L8 E/ a6 r, x# T  n/ |
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As shown by real-time PCR on RNA extracts, ClC-K1 and ClC-K2 mRNA were both found in DCT ( Fig. 3 ), a finding in agreement with previous results ( 19 ). The detection thresholds (crossing points) for ClC-K1, ClC-K2, and Pva were all in the same range and markedly higher than the threshold for NCC detection in the same extracts ( Fig. 3 A ). Because PCR efficiencies were higher for NCC, Pva, and ClC-K1 (1.9-2.0) than for ClC-K2 (1.70; Fig. 3 B ), these data suggested that NCC and ClC-K2 were expressed in the DCT to a greater extent than Pva or ClC-K1. In support of this conclusion, the relative expression of ClC-K2 in the entire DCT (calculated as described in METHODS ) was found to be about one-half that of NCC, whereas the levels of expression of ClC-K1 and Pva amounted to only 1% that of NCC, ( Fig. 3 C ). Despite these different levels of expression, under multiplex PCR conditions ( Fig. 3 D ), the detection thresholds for ClC-K1 and ClC-K2 were similar to each other (21 cycles PCR2), as in real-time PCR experiments, and higher than the threshold for Pva (17 cycles PCR2), the sequence exhibiting the lowest level of expression.
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Fig. 3. Expression of ClC-K1, ClC-K2, NCC, and Pva mRNA in DCT. A : number of PCR cycles necessary to reach the detection threshold [crossing points (CP)] in RT real-time PCR experiments on RNAs extracted from 0.1 mm of DCT. Results are expressed as means ± SE from 5 animals. **Significantly different from the other 3 groups at the P 0.001 level. *Significantly different from ClC-K1 ( P
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Single-Cell Molecular Analysis3 N! W, D3 d* w5 ~% Z1 m5 Y" E

Seventy-five cytosol samples collected from either DCT1 or DCT2 in 15 mice were analyzed for mRNA expression. Twelve samples (16%) in which no cDNA was detected and three others (4%) showing traces of Avpr2 genomic DNA were discarded.

Results obtained in the other samples (30 DCT1 and 30 DCT2 samples from experimental series I and II ) are reported in Table 2 and illustrated by representative examples ( Fig. 4 ). Because they were similar in both series, they were pooled when used to analyze the pattern of mRNA expression in DCT cells. The findings show that three samples collected from DCT2 (IC) could not be classified as DCT or CNT cells because they were devoid of NCC, Pva ( series II ), -ENaC ( series I ), and Avpr2. Samples like this, which expressed ClC-K2, but not ClC-K1 ( Fig. 4 D ), were found at a distance of 419-558 µm after the end of CTAL, suggesting that they may correspond to intercalated cells. All other DCT1 and DCT2 samples expressed NCC (or NCC and Pva in series II ) but neither NKCC2 nor Avpr2. Avpr2 colocalizing with KlK6 ( series III ) could however be detected in adjacent CNT cells ( Fig. 4 C ). -ENaC expression was found in most DCT2 samples (see below) but never in DCT1. Three different molecular phenotypes, common to DCT1 ( Fig. 4 A ) and DCT2 ( Fig. 4 B ) and identified as types 0, 1, and 2, could be defined on the basis of ClC-K expression in DCT cells (NCC-expressing cells). Type 0 was devoid of ClC-K and was found in similar proportions in DCT1 and DCT2 (37%). Type 1, which only expressed ClC-K2, was more abundant in DCT1 (53%) than in DCT2 (30%), and type 2, which expressed both ClC-Ks, was preferentially located in DCT2 (33 vs. 10% in DCT1). Becauseof this restricted localization of ClC-K1 mRNA, it is clear that the ClC-K1/ClC-K2 expression ratio in type 2 DCT2 cells is much higher than that calculated from experiments on whole DCT extracts ( Fig. 3 ).

Table 2. Single-cell RT-PCR analysis of mRNA expression in the DCT" I/ @4 _5 B8 N; H5 o1 ~

Fig. 4. RT-PCR analysis of ClC-K1 and ClC-K2 mRNA distribution in single DCT cells. A and B : representative electrophoresis gels illustrating the molecular phenotypes 1 and 2 found in DCT1 ( A ) and DCT2 cells ( B ) in 2 different experimental series ( series I : cDNA of NKCC2, NCC, Avpr2, -ENaC, ClC-K1, and ClC-K2 coamplified in the multiplex PCR1; series II : similar to series I except for Pva, which replaced NKCC2). Note that most DCT1 cells were of type 1 (ClC-K2 but no ClC-K1 mRNA expression), whereas types 1 and 2 (ClC-K1 colocalizing with ClC-K2 mRNAs) were both found in DCT2. C : data from experimental series III (NCC replaced by KlK, a marker of DCT and CNT cells) show that Avpr2 mRNA, when expressed (as is the case in CNT cells), could be detected by the single-cell approach. D : example of another molecular phenotype (IC) found in 3/30 late DCT2 cells. The absence of NCC and -ENaC (as well as of Avpr2 and Pva not shown here) suggests that this phenotype might correspond to intercalated cells.

Properties of the Cl - Channels

Separate patch-clamp experiments were used to find out whether DCT2 contains distinct Cl - channel types. Besides the cation channels, we recorded one other type of channel in cell-attached patches when we used PS in the pipette and bath ( Fig. 5 A ). The mean current-voltage relationship was linear ( Fig. 5 B ) and displayed a unit conductance of 8.4 ± 0.1 pS ( n = 13). The reversal potential ( E r = 0.5 ± 1.5 mV, n = 13) was close to zero. We qualitatively estimated the ionic selectivity by testing the effects on the reversal potential of changing the composition of the pipette solution. When NMDG-Cl (140 mM) was substituted for NaCl, there was no significant change in the reversal potential ( E r = -0.3 ± 1.8 mV, n = 6, not shown). In contrast, when 100 mM NaCl was replaced by Na gluconate, the reversal potential was shifted to the right by 26.7 ± 2.0 mV ( n = 4, not shown). This demonstrated that the 9-pS channels recorded in the cell-attached configuration were chloride selective.
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Fig. 5. Conductive properties of the 9-pS channel in the cell-attached configuration. A : mean single-channel current ( i )-voltage ( V c ) relationship from a membrane patch formed on a DCT immersed in physiological saline (PS). The pipette solution was PS. Each point is the average of 3-12 determinations from 13 separate patches. SE is shown as error bars when larger than symbols. B : representative current recordings at various clamped potentials ( V c, as indicated on the right of each trace). *One-half-amplitude current substate is apparent at -60 mV.$ q: k( }9 L! k

We ascertained the anion/cation selectivity of the channel in the excised mode. With PS on both sides of the membrane patch, the conductance ( g = 8.8 ± 0.6 pS, n = 10) was not statistically different from that in cell-attached mode. The reversal potential was shifted from 0.6 ± 0.9 mV ( n = 10) to -36.8 ± 2.8 mV ( n = 9) when the bath solution was replaced by a low-NaCl solution (not shown), indicating anion selectivity. We calculated a P Na /P Cl value of 0.09 ± 0.02 ( n = 9).

We then studied in detail the permeability to Br -, NO 3 -, and I - in the DCT2. To our surprise, although we had detected only one class of conductance ( 9 pS), the relative permeabilities fell into two divergent groups, with values below and above unity, respectively. Figure 6 shows the corresponding current-voltage relationships for the three ions tested; the relative permeabilities are plotted in Fig. 7 A (DCT2 columns). In 12 cases, we were able to determine the relative permeability for Br - and NO 3 - in the same patches ( Fig. 7 B, ): in each case, a low permeability (i.e., less than unity) for Br - correlated with low permeability for NO 3 - and a high permeability for Br - correlated with high permeability for NO 3 -. Similarly, channels with low permeability to NO 3 - had also permeability to I - of less than unity ( n = 5, not shown), and one patch with an NO 3 - permeability higher than unity (1.32) had an I - permeability of 2.4 (not shown). These findings show that the DCT2 contains two types of Cl - channels with similar unit conductance but very different anion permeability sequences (see Table 3 for numerical data). The channel with the anion selectivity sequence I - NO 3 - Br - Cl - (type B, 29% of patches) was less frequent than that displaying the sequence Cl - I - = NO 3 - = Br - (type A, 71% of patches, total number of patches: 24). As illustrated in Fig. 8, these two channels cannot be distinguished by visual inspection, because they have a very similar "signature."

Fig. 6. Anion selectivity of the Cl - channels. The two types of current i - V c relationships obtained in inside-out patches when 130 mM NaCl was replaced by NaBr ( A ), NaNO 3 ( B ), or NaI ( C ) are shown on the left together with excerpts of recordings on the right. The pipette contained PS. Circles are used to designate type A channels are used and triangles are used for type B channels. Data from DCT1 and DCT2 were pooled and are shown either as means ± SE of 3-27 patches (filled symbols) or represent 1-2 measurements (open symbols). Single-channel openings were rarely resolved at negative voltages for iodide and between -20 to -70 mV for nitrate in the case of the type A channel. Accordingly, * indicate current traces with no clear channel openings. In the case of the type B channel in the presence of iodide, 2 individual measurements are shown. The lines are fits with the Goldman-Hodgkin-Katz equation.

Fig. 7. Anion selectivity of the Cl - channels. A : histograms showing the relative permeabilities of the Cl - channels for bromide, nitrate, and iodide investigated in the DCT1 and DCT2. B : histogram showing individual measurements of permeability to nitrate plotted against permeability to bromide for DCT1 ( ) and DCT2 ( ) data. Experimental points aggregate within one region in which both permeabilities are less than unity (type A), and another where both permeabilities are greater than unity (type B).( X. F7 W) B+ v$ a3 n$ `8 d

Table 3. Anion selectivity of Cl - channels in the DCT

Fig. 8. Channel current recordings for type A ( top trace) and type B ( bottom trace) Cl - channels. The 2 recordings are indistinguishable. The recording was done in the inside-out configuration, with low-NaCl solution on the inner side of the membrane patch and PS in the pipette.( b- L) m& S: S* L

In a previous study ( 19 ), we reported one Cl - channel in the DCT1 (corresponding to the present type A channel), which we suggested might be a ClC-K2 channel. Given the results obtained for DCT2, we reexamined the anion selectivity of the 9-pS Cl - channels in DCT1. The results are shown in Fig. 7 A (DCT1 columns) and Fig. 7 B ( ); they indicate that type A and type B channels are both present in DCT1 (see Table 3 ). However, the frequency of the type B channel is much lower (13% of patches, total number of patches: 15) than that of the type A channel (87% of patches).# ^6 Q9 K4 T8 Y% D6 _1 ], e7 H

The type A Cl - channel in DCT1 is sensitive to intracellular pH ( 19 ). We investigated the effects of the intracellular pH on inside-out patches formed on DCT2 cells after checking the anion selectivity. We observed that the activity of type A Cl - channels decreased with pH ( n = 4; Fig. 9 ). In one case, we were able to confirm that the type B Cl - channel was also inhibited at an acidic pH (not shown).
  p" u  |! T6 c/ d+ U0 `
Fig. 9. Sensitivity of type A Cl - channel toward intracellular pH (pH i ). A : channel current traces showing that the activity of the type A channel decreased at acidic pH. The recording was done in the inside-out configuration, with low-NaCl solution on the inner side of the membrane patch and PS in the pipette. The value of pH i is indicated on the right of the tracings. B : histogram for the effects of pH i on the open channel probability ( NP o ). Each bar is the mean of 4 measurements.

DISCUSSION

Single-Cell PCR Approach in DCT Cells
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Single-cell RT-PCR studies have already been carried out in a variety of tissues ( 12, 14, 29 ). Among them, the technique developed by Rossier for studying motoneurones ( 1 ) appeared particularly suitable for analyzing heterogeneous tissues, because it can be used to coamplify different cDNA species from the same sample and, thereby, to identify the cell type. We tentatively applied this approach to DCT to study the cellular localization of ClC-K1 and ClC-K2 mRNA.
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Ninety-five percent of the samples showing cDNA (30/30 DCT1 and 27/30 DCT2; Table 2 ) had the molecular phenotype expected from DCT cells, expressing NCC and, when tested, Pva ( 6, 17 ), but not expressing either NKCC2 ( 16, 26 ) or Avpr2 ( 20 ). The remaining samples that had been collected from the terminal portion of DCT2 did not correspond to either DCT (no expression of NCC, -ENaC, or Pva) or principal CNT cells (no expression of KlK6, Avpr2, or -ENaC; see Fig. 4 D ). These samples, which expressed ClC-K2, corresponded to 10% of the total number of DCT2 cells. Because they were also found in CNT (results not shown), they likely correspond to intercalated cells, but no definite conclusion can be drawn until tests for specific markers have been performed." ^4 F, P5 w$ G8 P# U4 [6 c

As expected from immunofluorescence studies ( 17 ), -ENaC was found colocalized with NCC in most DCT2 cells, but never in DCT1. In contrast, Pva mRNA was found throughout the length of the DCT, indicating that Pva cannot be used as a specific marker of DCT1 cells in PCR experiments. However, because the presence of mRNa does not necessarily mean that the corresponding protein is translated, our data do not allow us to conclude whether Pva is evenly distributed along DCT, as described by Campean et al. ( 6 ), or specifically localized in DCT1, as reported by Loffing et al. ( 17 ).& m- r- B& T3 d  `% F# f+ e; l: m
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Interestingly, the DCT2 cells that did not display -ENaC mRNA were those of type 0 that express only NCC and Pva ( Table 2 ). This raises the question of whether type 0 cells are a real cellular subtype or an artifact resulting from some technical problem impairing cDNA detection. In our opinion, this latter hypothesis is unlikely because Pva mRNA was detected in all the samples, even though it has a low level of expression in DCT (only 1% of NCC). This reproducibility also shows that heterogeneity of cytosol sampling did not result in scatter under our experimental conditions. Taken together, these data indicate that the single-cell RT-PCR approach developed by Rossier's group is a reliable tool for analyzing the molecular phenotype of cell types present in the distal heterogeneous nephron segments." F+ }# |2 M6 M, p, x
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ClC-K1 and ClC-K2 mRNA Expression in DCT Cells
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PCR analysis at the single-cell level gave an interesting insight into the distribution of ClC-K1 and ClC-K2 mRNA along this part of the renal tubule. A first unexpected finding was the existence of 37% DCT cells in which ClC-K mRNA was not detected (type 0). Assuming that these type 0 cells are involved in the transcellular reabsorption of chloride, we propose that basolateral Cl - transport must involve some other pathway, such as an electroneutral KCl cotransporter ( 4, 34 ) or a non-ClC Cl - channel.( ]) e/ y* E: g) q) R4 w' @
3 M" M2 _* @( O
Single-cell PCR experiments also showed that more cells express ClC-K2 than ClC-K1. Indeed, 65% of the cells analyzed along the entire DCT contain ClC-K2 mRNA vs. only 20% containing ClC-K1. Moreover, the two channels have differing cell distribution patterns. ClC-K2 mRNA is evenly distributed along the DCT (63% of the cells in DCT1 and 67% in DCT2); in contrast, ClC-K1 mRNA, although present in a minor subset of DCT1 cells (10%), is preferentially located in DCT2 (33%), always colocalizing with ClC-K2 (type 2). To summarize, besides type 0, most of the cells in DCT1 express only ClC-K2 mRNA (type 1). In contrast, one in two DCT2 cells coexpresses ClC-K1 and ClC-K2 mRNA, making it possible for ClC-K1/ClC-K2 heterodimers to be formed.9 K3 M" }7 V. _& A

Candidate ClC-K Cl - Channel in DCT26 {* \9 v1 q  d
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Due to the paucity of information so far provided by heterologous expression studies, it is not easy to compare the macroscopic data obtained from ClC-K cloned channels with the single-channel currents obtained here in mouse DCT cells. In particular, the unit conductance of the human ClC-Ks coexpressed with barttin in X. laevis oocytes has not been determined. Moreover, both ClC-Ks produce higher currents when the extracellular pH or calcium is elevated, and they have only slightly differing anion permeability sequences: Cl - NO 3 - = Br for ClC-Kb (K2) and Cl - NO 3 - Br - for ClC-Ka (K1) ( 8, 35, 36 ).
. C. n$ e, H1 f' p/ r4 I
There is a 9-pS Cl - channel in the DCT1 basolateral membrane exhibiting the anion permeability sequence Cl - I - NO 3 - = Br -, which is inhibited at an acidic intracellular pH, and has a higher open probability when the extracellular pH or calcium is elevated ( 19 ). The type A Cl - channel found in DCT1 and DCT2 throughout the present study has similar properties in terms of conductance, anion permeability sequence, and sensitivity to intracellular pH. On the grounds of these properties and the single-cell RT-PCR results, we can reasonably conclude that these two channels constitute a single functional type of channel, constituted by ClC-K2 in type 1 cells, i.e., in 53 and 30% of DCT1 and DCT2 cells, respectively.

Despite the presence of ClC-K1 mRNA in 33% of DCT2 cells (type 2), we were unable to record channels displaying the exact permeability sequence of the human ClC-Ka ( 8, 36 ). In particular, we never recorded the 40-pS Cl - channel, previously described in the CTAL, which displays this sequence ( 22 ), but has not been identified at the molecular level. It should, however, be noted that the permeability sequence (not determined for the mouse) differs between the two ClC-Ks only for bromide [0.8 for rat ClC-K1 and human ClC-Ka, 0.3 for human ClC-Kb, ( 8, 36 )]. If ClC-K1 and ClC-K2 have comparable unit conductance (which remains to be demonstrated), ClC-K1 may have been masked by ClC-K2 channels that are more numerous in DCT. In addition, because ClC-K1 mRNA was never found alone, but always coexpressed with ClC-K2, it is also possible that the type A channel corresponds to not only ClC-K2 and/or ClC-K1 homodimers but also ClC-K1/ClC-K2 heterodimers or a mixture of all combinations. Alternatively, we cannot exclude the possibility that the ClC-K1 protein is either absent or electrically silent in the normal animal and that protein translation or activation is triggered by some unidentified physiological condition.4 C6 |* [4 h0 \( S) C: W
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A Second Cl - Channel with Indeterminate Molecular Identity

The selectivity experiments revealed another Cl - channel (type B) with contrasting anion selectivity but confusingly similar conductance. We previously described a similar channel in the basolateral membrane of mouse CTAL ( 10 ), where both ClC-K1 and ClC-K2 mRNAs are expressed ( 33 ). Although the distribution of the type B channel (29% of DCT2 vs. 13% of DCT1 patches) matches that of ClC-K1 mRNA (33% of DCT2 vs. 10% of DCT1 measurements), it seems unlikely that the type B channel actually corresponds to the ClC-K1/ClC-K2 heterodimer or ClC-K1 homodimer that we were trying to detect in DCT2. Indeed, the NO 3 - Br - Cl - permeability sequence contrasts sharply with that of the ClC-Ks (Cl - Br - NO 3 - ) and other ClCs ( 13, 31 ) and is more similar to that of the calcium-dependent and volume-activated Cl - channels ( 13 ). At present, the molecular identity of volume-activated Cl - channels is highly controversial ( 13 ). Moreover, our experiments in the presence of EGTA indicate that the activity of the type B Cl - channel is not strictly dependent on calcium. This apparently rules out the possibility that this channel could belong to any of the cloned families of calcium-dependent Cl - channels [i.e., bestrophin and CLCA ( 9, 24, 28, 30 )]. Thus further investigation is called for before the type B channel can be characterized at the molecular level.& A( @* h6 S7 @# k( _& v" m

Major but not Exclusive Role for ClC-K2 in the Process of Cl - Absorption* R& R8 N6 m! r+ }6 Z
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Our results suggest the major implication of ClC-K2 in the process of Cl - transport throughout the DCT, and the possible participation of ClC-K1, especially in DCT2. However, even though the role of ClC-Ks is predominant, it cannot be exclusive for two reasons. First, 37% of DCT1 and DCT2 cells (i.e., those expressing mRNA for the Na-Cl cotransporter) do not express any ClC-K mRNA (type 0 cells). Cl - ions that enter type 0 cells at the apical membrane are thus likely to leave from the basolateral side through a pathway distinct from ClCKs, for instance via a KCl cotransporter ( 4, 34 ) or another Cl - channel. Second, the non-ClC, type B Cl - channel, which is located at the basolateral membrane, is also in a position to participate in Cl - absorption across DCT2. Because its molecular identity is unknown and its conductance similar to that of the ClC-K channel, it is not possible to determine the cell type in which it is located.4 ^9 ?# F$ t& ^3 _/ P  H( e
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To conclude, single-cell PCR and patch-clamp experiments have shown that the functional heterogeneity between DCT1 and DCT2 includes the expression of Cl - channels. On the basis of ClC-Ks distribution patterns, they have also revealed the presence of several different cell types in each subsegment. This cellular diversity may be necessary to ensure the independent regulation of the various transport processes that take place in the DCT. Alternatively, it may help to maintain DCT functions when the activity of any of these chloride channels is impaired., W' }4 p% ]3 B" E! ~. [/ l% w

GRANTS
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A. Nissant and S. Lourdel hold PhD fellowships from the Ministère de la Recherche; M. Paulais is an Institut National de la Santé et de la Recherche Médicale researcher, and M. Imbert-Teboul is a Collège de France researcher.

ACKNOWLEDGMENTS, F! Z% E! w3 G+ Z6 L
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We are greatly indebted to J. M. Elalouf for helpful advice and discussion. The English text has been revised by M. Ghosh.

Present address of P. Marvao: Dept. of Physiology, University of Lisbon, 1169-056 Lisbon, Portugal.

Present address of S. Lourdel: Zentrum für molekulare Neurobiologie, University of Hamburg, 20251 Hamburg, Germany.

Present address of S. Baillet: Service de Nephrologie, Centre Hospitalier Universitaire 38043 Grenoble, France.
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